Electroacupuncture Stimulation Alleviates Inflammatory Pain in Male Rats by Suppressing Oxidative Stress.

In the present study, we focused on whether the analgesic effect of Electroacupuncture (EA) is related to the regulation of oxidative stress. We established a chronic inflammatory pain model in male rats by a single injection of complete Freund's adjuvant (CFA) and then treated the animals with daily EA stimulation at the site of "zusanli". The analgesic effect of EA was evaluated by measuring the paw withdrawal threshold (PWT) when rats received mechanical and thermal pain stimulation. The levels of inflammation-related molecules and oxidative stress-related markers in the spinal cord were measured by western blotting or ELISA kits. EA stimulation and antioxidants effectively increased the PWT in CFA rats. Co-treatment of CFA rats with the ROS donor t-butyl hydroperoxide (t-BOOH) further decreased the PWT and weakened the analgesic effect of EA. EA treatment inhibited inflammation and oxidative stress, as shown by decreased levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and MDA and increased activity of SOD and catalase. Moreover, EA reduced the expression of p-p38, p-ERK, and p-p65 and simultaneously downregulated the expression of TRPV1 and TRPV4 in CFA rats. In an in vitro study, direct stimulation with t-BOOH to the C6 cells increased the production of TNF-alpha, IL-1beta, IL-6, activated p38, ERK, and p65 and up-regulated the expression of TRPV1 and TRPV4, and these effects could be prevented by the ROS scavenger PBN. Taken together, our data indicate that the inhibition of oxidative stress and the generation of ROS contribute to the analgesic effect of EA in male CFA rats.

[Salvianolate protects H9c2 cells from hypoxia/reoxygenation injury-induced apoptosis by attenuating mitochondrial DNA oxidative damage].

Objective: To investigate the possible mechanism related to the protective effects of salvianolate in H9c2 cells underwent hypoxia/reoxygenation (H/R)-injury. Methods: H9c2 cells were divided into four groups: control group, salvianolate group (S group), H/R group, and salvianolate+ H/R group(S+ H/R group), in which the H9c2 cells were pretreated with salvianolate before H/R-treatment.Apoptotic cells were detected by Tunel assays and AnnexinⅤ-FITC apoptosis detection kit.The intracellular ATP level, the change of mitochondrial membrane potential and the mitochondrial DNA oxidative damage were also determined in these groups. Results: (1) The apoptosis rate of H/R group(26.36±5.14)% was significantly higher compared to control group(2.71±1.66)%(P=0.000 4), which could be significantly reduced in S+ H/R group(17.28±4.75)%(P=0.012 8 vs. H/R group , P=0.003 9 vs. control group). The ratio of AnnexinⅤ and PI double positive cells in H/R group(28.23±6.73)% was significantly higher compared to control group(3.53±2.83)%(P=0.001 1), which was significantly reduced in S+ H/R group(18.10±4.56)%(P=0.037 2 vs. H/R group, P=0.038 3 vs. control group). (2)The ATP level of H9c2 cells in H/R group(49.05±10.12)% was significantly lower than in control group 100%(P=0.000 5), which was significantly increased in S+ H/R group(68.67±13.32)%(P=0.019 9 vs. H/R group). Confocal microscope showed that red fluorescence was dominant in the control group, red fluorescence was significantly reduced, while green fluorescence was significantly increased in H9c2 cells of H/R group and the fluorescence ratio of red to green in H/R group((37.13±8.47)%) was significantly decreased compared to control group (100%, P=0.000 1), fluorescence ratio of red to green was significantly increased in S+ H/R group((63.77±12.32)% vs. H/R group, P=0.007 3). (3)The mitochondrial DNA oxidative damage in different groups: there was only few 8-hydroxyguanine (8-OHdG) expression, which marked as green, in control group, and 8-OHdG expression was significantly upregulated in H/R group, moreover, the 8-OHdG was co-localized with mitochondria.The expression of 8-OHdG was significantly lower in S+ H/R group compared to H/R group. Conclusion: Salvianolate can reduce mitochondrial DNA oxidative damage, and protect mitochondrial function, thus inhibit myocardial cell apoptosis and eventually reduce the myocardial H/R-injury in H9c2 cells.

[The effects and related mechanism of salvianolate on rats with myocardial ischemia-reperfusion injury].

Objective: To investigate the effect and related mechanism of salvianolate on myocardial ischemia-reperfusion (I/R) injury in rats. Methods: Thirty-six adult Sprague-Dawley rats were divided into three groups (n=12 each) using random number table method: control group (coronary artery was not ligated) , I/R group (myocardial I/R model was established by ligation and opening of left anterior descending coronary artery) , and salvianolate+I/R group (5 mg/kg of salvianolate was injected through the tail vein at the time of reperfusion) .Triphenyltetrazolium chloride (TTC) stain was utilized to measure the myocardial infarct size. The ELISA method was used to detect myocardial necrotic markers, including cardiac troponin T (TnT) , creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) . Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to analyses the levels of apoptosis. The levels of cleaved Caspase-3 protein were analyzed with Western blot.Cold luminescence method was used to detect the ATP level of myocardial tissue. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in myocardial tissue were detected by immunofluorescence. Results: (1) The infarct size in control group, I/R group and salvianolate+I/R group were 0, (23.90±5.66) %, and (12.06±5.97) %, respectively (P<0.01 or 0.05) . (2) The TnT level was (0.04±0.03) , (16.96±2.80) , and (6.95±2.31) ng/ml, the CK-MB level was (43.6±23.5) , (1 135.8±180.6) ,and (390.3±121.5) U/L, the LDH level was (119.0±58.6) , (1 838.6±543.8) , and (631.6±228.3) U/L in control group, I/R group and salvianolate+I/R group, all significantly lower in salvianolate+I/R group than in I/R group (all P<0.01) . (3) The rate of TUNEL positive myocardial cells were (1.07±1.16) %, (21.36±4.11) %,and (13.30±3.67) % in control group, I/R group,and salvianolate+I/R group (all P<0.01) . (4) The cleaved Caspase-3 expression was 0.11±0.05, 0.84±0.20,and 0.43±0.09 in control group, I/R group, and salvianolate+I/R group (all P<0.01) . (5) The ATP level of myocardial tissue was (100.0±0.0) %, (34.2±9.2) %,and (62.1±18.0) % respectively in control group, I/R group, and salvianolate+I/R group (all P<0.01) . (6) There was almost no 8-OHdG expression in the myocardial tissue of control group. The expression of 8-OHdG in the myocardial tissue of I/R group was greater than that of the control group. The expression of 8-OHdG in the myocardial tissue of salvianolate+I/R group was less than that of the I/R group. Conclusion: Salvianolate may alleviate myocardial I/R injury of rat through reducing the mitochondrial DNA oxidative damage, protecting mitochondrial function and inhibiting the apoptosis of myocardial cells.

A laccase with antiproliferative activity against tumor cells from an edible mushroom, white common Agrocybe cylindracea.

A laccase, with HIV-1 reverse transcriptase inhibitory activity (IC(50)=12.7 µM) and antiproliferative activity against HepG2 cells (IC(50)=5.6 µM) and MCF7 cells (IC(50)=6.5 µM), was purified from fresh fruiting bodies of the edible white common Agrocybe cylindracea mushroom. The laccase, which had a novel N-terminal sequence, displayed a molecular mass of 58 kDa within the range reported for most other mushroom laccases. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, SP-Sepharose, and Q-Sepharose and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but unadsorbed on SP-Sepharose. Its optimum pH was pH 3-4 and its optimum temperature was 50°C. The activity of the isolated laccase differed from one substrate to another. The ranking was ABTS>N,N-dimethyl-1,4-phenylenediamine>hydroquinone>catechol>2-methylcatechol>pyrogallol.

[The structure identification of Julibroside J6 from Albizia julibrissin Durazz].

OBJECTIVE: To study the saponin from Albizia julibrissin. METHODS: A saponin was separated by using chromatography and its structure was elucidated on the basis of spectral data. RESULTS: A saponin was obtained and it's structure was identified as 3-O-[beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)-beta- D-2-deoxy-2-acetamidoglucopyranosyl]-21-O-(6S-2-trans-2-hydroxymethyl- 6-methyl-6-O-[4-O-(6S-2-trans-2-hydroxymethyl-6-methyl-6-hydroxy-2, 7-octadienoyl)-beta-D-quinovopyranosyl]-2, 7-octadienoyl)-acacic acid-28-O-beta-D-glucopyranosyl(1-->3)-[alpha-L-arabinofuranosyl- (1-->4)]-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl ester. CONCLUSION: The saponin is a new compound named Julibroside J6.

[Structure determination of three saponins from the stem bark of Albizzia julibrissin Durazz].

Three new saponins named Julibroside J1, J2 and J3 were isolated from the stem bark of Albizzia julibrissin Durazz (Albizziae Cortex). Based on chemical and spectral methods, e.g. 1H- and 13C-NMR, DEPT, COSY CH-COSY, TOCSY, HMQC-COSY, HMQC-TOCSY, NOESY, HMBC, their structures have been identified as; Julibroside J1 (one triterpene, nine sugars, two monoterpenes I); Julibroside J2(one triterpene, eight sugars two monoterpenes II); Julibroside J3(one triterpene, nine sugars two monoterpenes III).

[Studies on the triterpene sapogenins from Albizziae Cortex].

A new sapogenin named Julibrogenin A was isolated from the stem bark of Albizzia julibrissin Durazz (Albizziae Cortex). Based on chemical and spectral studies, their structure has been identified as 21-O-(2-hydroxymethyl-6-methyl-6-methoxyl-2,7-octadienoyl)-acacic acid. In addition, machaerinic acid lactone, machaerinic acid methylester, acacigenin B and julibrotriterpenoidal lactone A were also isolated from this plant and characterised.

[Two new flavanone glycosides from Glycyrrhizia inflata].

Two new flavanone glycosides having two chains of sugar moeity were isolated from the roots of Glycyrrhizia inflata Bat by repeated CC and HPLC. They were identified to be liquiritigenin-7-O-beta-D-apiofuranosyl-4'-O-beta-D-glucopyranoside(I) and liquiritigenin-7-O-beta-D-(3-O-acetyl)-apiofuranosyl-4'-O-beta-D- glucopyranoside (II) on the basis of chemical and spectroscopic methods.

Flavonoids from Epimedium wanshanense.

A novel flavonol glycoside named wanepimedoside A was isolated from the whole plant of Epimedium wanshanense, along with fifteen known flavonoids, anhydroicaritin, desmethylanhydroicaritin, icarisid I and II, quercetin, ikarisoside A and B, sagittatoside B, 2"-O-rhamnosylicarisid II, icariin, 2"-O-rhamnosylikarisoside A, epimedin B, epimedin C, and diphylloside A and B.

Triterpenoid saponins from Bupleurum smithii var. parvifolium.

Four triterpenoidal saponins, prosaikogenin A and saikosaponins b1, n and o, were isolated from the roots of the title plant for the first time. Saikosaponin O is a new compound, which was identified as 3 beta,16 beta,23,28-tetrahydroxyolean-11,12(18)-diene-3-O-beta-D-glucopy ran osyl-(1-->2)-beta-D-glucopyranosyl-(1--> 6)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranoside.

Flavonol glycosides from Epimedium koreanum.

A novel flavonol glycoside named caohuoside-B was isolated from the aerial parts of Epimedium koreanum, along with a known flavonol glycoside, epimedokoreanoside-I. Their structures were established by spectroscopic methods. The new compound was elucidated as anhydroicaritin 3-O-beta-D-(4,6-O- diacetyl)glucopyranosyl-(1-->3)-alpha-L-(4"-O-acetylrhamnopyranoside+ ++)-7-O-beta- D-glucopyranoside.

[Isolation and identification of two new saponins from Bupleurum smithii Wolff].

Two new saikosaponins of oleanane type were isolated from the roots of Bupleurum smithii Wolff, collected in Minhe County, Qinghai, namely saikosaponin k (VIb) and saikosaponin 1 (VIII). Their structures were elucidated on the basis of spectral analysis of UV, IR, 1HNMR, 13CNMR and FAB-MS. Saikosaponin k (VIb) was identified as 3 beta, 16 beta, 23,28-tetrahydroxyoleana-11,13(18)-dien-3-O-beta-D-xylopyranosyl-(1-->2)-beta- D-glucopyranosyl-(1-->3)-beta-D-fucopyranoside, and saikosaponin 1 (VIII) as 3 beta, 16 alpha, 23,28,30-pentahydroxyoleana-11,13(18)-dien-3-O-beta-D- glucopyranosyl-(1-->3)-beta-D-fucopyranoside, respectively.

[Studies on the triterpenoids from roots of Glycyrrhiza squamulosa Franch].

A new triterpenoid, squasapogenol (S-11), was isolated from the hydrolytic products of dilute ethanol (10%) extract of the roots of Glycyrrhiza squamulosa Franch, along with four known triterpenoids. The structure of S-11 was elucidated as olean-11,13(18)-diene-3 beta, 22 beta-diol on the basis of chemical and spectroscopic data. The four known compounds were identified as betulinic acid(S-8), methyl ester of macedonic acid(S-9), soyasapogenol B (S-10) and olean-13(18)-ene-22 alpha-Cl-3 beta, 24-diol(S-12).

[Studies on the chemical constituents of Glycyrrhiza pallidiflora Maxim].

A species of the genus Glycyrrhiza, G. pallidiflora Maxim, growing in Jiangsu and Hebei Provices of China, has been little studied before on its chemical constituents. This paper reports the isolation and chemical elucidation of five compounds from this plant, one of them is a new compound named glypallichalcone (P-2). Their chemical structures were elucidated by means of chemical and spectrometric analysis (UV, IR, MS, 1HNMR and 13CNMR) and were first reported to be present in this plant. Glypallichalcone (P-2), was found to be 4-hydroxy-2,4'-dimethoxy-chalcone. The known compounds were identified to be 4'-O-methylcoumestrol (P-1), N-acetyl-glutamic acid (P-3), formononetin and beta-sitosterol (P-5).

Glyyunnansapogenins G and H: Two New Pentacyclic Triterpenoids of the 18alphaH-Oleana-9(11),12-diene Type from Glycyrrhiza yunnanensis Roots.

Two new pentacyclic triterpenoid sapogenins of the 18alpha H-oleana-9(11),12-diene type named glyyunnansapogenins G and H were isolated from the roots of GLYCYRRHIZA YUNNANENSIS Cheng f. et L. K. Tai. Their structures were elucidated as 18alpha H-oleana-9(11),12-diene-3beta,21alpha,29-triol and 18alpha H-3beta,21alpha-dihydroxyoleana-9(11),12-dien-29-oic acid, respectively, by spectroscopic methods. This is the first report of the occurrence of the 18alpha H series of oleana-9(11),12-diene compounds in nature.

[Quality evaluation of Chinese licorice].

The contents of 12 compounds, viz. glycyrrhizinic acid, uralsaponin A, uralsaponin B, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycycoumarin, isoglycycoumarin, licochalcone A, glycyrol and isoglycyrol, present in Chinese licorice, the roots and rhizomes of 8 Glycyrrhiza species collected from 15 districts in China, were determined by high performance liquid chromatographic method. The quality evaluation of Chinese licorice was discussed according to the results of the determinations.

[The structure of glyeurysaponin].

A species of the genus Glycyrrhiza, G. eurycarpa P.C. Li recently reported as a new species growing in Gansu Province Xinjiang Autonomous Region has rarely been studied before on its chemical constituents. This paper reports the isolation and chemical elucidation of two triterpene glucosides named glyeurysaponin (K-4) and uralsaponin B (K-3) from this species collected in Jinta County, Gansu Province. Their chemical structures were elucidated by means of chemical and spectrometric analysis (UV, IR, MS, 1HNMR and 13CNMR) and were first reported to be present in this plant. Glyeurysaponin (K-4), C42H62O16 was obtained as white crystals, mp 288 degrees C (d), [alpha]18D + 22.5 degrees (c 0.62,60%MeOH). Its structure was found to be be 3 beta-hydroxy-11-oxo-olean-12-en-30-oic acid-3-O-beta-D-glucuronopyranosyl-(1----4)-beta-D-glucuronopyr anoside. Glyeurysaponin is a new triterpenoid saponin. The known saponin was identified to be 3 beta-hydroxy-11-oxo-olean-12-en-30-oic acid-3-O-beta-D-glucuronopyranosyl-(1----3)-beta-D-glucuronopyr anoside, uralsaponin B.

[Separation and quantitative determination of three saponins in licorice root by high performance liquid chromatography].

A new analytical method for the separation and simultaneous determination of three saponins in Licorice root by high performance liquid chromatography has been developed. The saponins, glycyrrhizic acid (S-I), uralsaponin B (S-II) and uralsaponin A (S-III) were separated and determined by use of the solvent system CH3CN-3% HOAc (H2O) (47:53) at 248 nm, and with gradient increasing flow rate, the operation can be completed in 20 min. This method is sensitive and acuate with good reproducibility. The contents of three saponins in Chinese Licorice roots derived from four Glycyrrhiza species were determined.

[Chemical studies of coumarins from Glycyrrhiza uralensis Fisch].

This paper reports the isolation and identification of eight crystalline substances (I, II, III, IV, V, VI, VII, VIII) from the root of Glycyrrhiza uralensis Fisch. Besides the known compounds liquiritin, hexacosance, beta-sitosterol, licoricone liquiritigenin and etc, a new constituent, named neoglycyrol was obtained by sillica gel and polyamide column chromatographic method. Its chemical structure was elucidated by means of chemical and spectrometric analysis (UV, IR, NMR and MS). Neoglycyrol, C21H18O6, yellow needles with mp 263.5-265 degrees C, possesses one methoxyl, one isopentenyl and two hydroxyls. Its diacetate derivative is C25H22O8 with mp 202-203.5 degrees C. Its dimethyl ether derivative is C23H22O6 with mp 207-208.5 degrees C. Its structure was found to be VII.

[Glyyunnanprosapogenin and glyyunnansapogenin from the roots of Glycyrrhiza yunnanensis].

Two new triterpenoidal sapogenins of oleanane type, namely glyyunnanprosapogenin D (V) and glyyunnansapogenin F (IX) were isolated from the roots of Glycyrrhiza yunnanensis Cheng f. et L. K. Tai, family Leguminosae, collected from Yunnan Province, China. V was obtained as hexaacetate and dimethyl ester form (Vc), mp 168-170 degrees C, C56H78O22, which gave an aglycone macedonic acid (V') and two molecules of glucuronic acid after hydrolysis with hydrochloric acid. The spectral analysis of 13CNMR and 1HNMR showed that Vc had two glycoside residues attached to the aglycone, and therefore the structure of V was established as oleana-11, 13(18)-dien-29-oic acid, 3 beta, 21 alpha-di-(O-beta-D-glucuronic acid) pyranoside. IX was obtained as its diacetate and monomethyl ester form (IXc), mp 188-190 degrees C, C35H50O7. The IR, 1HNMR and UV spectra showed that the IXc had a skeleton of oleanane type triterpenoid with heteroannular diene. IXb, diacetate of IX, unpurified, was converted to IXf with chromium trioxide in glacial acetic acid. IXf, mp 160-164 degrees C, C30H46O8, was proved to be olean-12-en-11-oxo-3 beta,24-diacetoxy-18 beta-hydroxy-30-oic acid, 30 in equilibrium 18 beta lactone with another carbonyl group at C-15,16,21 or 22 by the analysis of 13CNMR, 1HNMR and MS spectra.(ABSTRACT TRUNCATED AT 250 WORDS)